Background The paired-domain transcription factor paired box gene 6 (PAX6) causes a wide spectrum of ocular developmental anomalies, including congenital aniridia, Peters anomaly and microphthalmia. Here, we aimed to functionally assess the involvement of seven potentially non-canonical splicing variants on missplicing of exon 6, which represents the main hotspot region for loss-of-function PAX6 variants.
Methods By locus-specific analysis of PAX6 using Sanger and/or targeted next-generation sequencing, we screened a Spanish cohort of 106 patients with PAX6-related diseases. Functional splicing assays were performed by in vitro minigene approaches or directly in RNA from patient-derived lymphocytes cell line, when available.
Results Five out seven variants, including three synonymous changes, one small exonic deletion and one non-canonical splice variant, showed anomalous splicing patterns yielding partial exon skipping and/or elongation.
Conclusion We describe new spliceogenic mechanisms for PAX6 variants mediated by creating or strengthening five different cryptic donor sites at exon 6. Our work revealed that the activation of cryptic PAX6 splicing sites seems to be a recurrent and underestimated cause of aniridia. Our findings pointed out the importance of functional assessment of apparently silent PAX6 variants to uncover hidden genetic alterations and to improve variant interpretation for genetic counselling in aniridia.
- genetic screening/counselling
- molecular genetics
- vision research
Data availability statement
All data relevant to the study are included in the article or uploaded as supplemental information.
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Contributors All the authors have substantially contributed to design, data acquisition, analysis or interpretation as well as to draft or revise the work and have approved the manuscript.
Funding This work was supported by several research grants from Spanish Institute of Health Carlos III (ISCIII) and the European Regional Development Fund (ERDF): PI17/01164, awarded to MC and the Spanish Centre for Biomedical Network Research on Rare Diseases (CIBERER, 06/07/0036 grant), awarded to CA; from the Ministry of Economy and Competitiveness/ERDF (MINECO, SAF2013-46943-R), the Spanish Mutua Madrileña Foundation and Spanish Foundation of Rare Diseases (FEDER), awarded to MC, as well as the Regional Government of Madrid (CAM, B2017/BMD3721), the University Chair UAM-IIS-FJD of Genomic Medicine and the Ramon Areces Foundation, awarded to CA. MC was sponsored by the ISCIII Miguel Servet Programme (CPII17_00006) and MT received a PhD fellowship from Conchita Rábago Foundation. JM and AT were sponsored by Regional Government of Madrid (CAM, PEJ-2017-AI/BMD-7256 and PEJD-2018-PRE/BMD-9453).
Competing interests None declared.
Provenance and peer review Not commissioned; externally peer reviewed.
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